Method and apparatus for analysis of particles in a liquid sample

Abstract

The invention relates to methods for analysis of particles in a liquid sample, the sample being retained in a sample retaining device, the method comprising: acquiring, by means of an image acquiring device, a plurality of images of said sample at different focus planes within the sample retaining device; analysing said images, by means of an image analyser, for identifying which, if any, of the particles of the sample are imaged in focus in each of the images, and analysing those particles which have been identified as being imaged in focus; wherein said plurality of images are acquired at different, essentially parallel, focus planes, which planes are separated from each other by a distance, said distance being less than 10 micrometers. The invention also relates to apparatuses adapted to be used for the inventive methods.

1. A method for analysis of particles in a liquid sample, the sample being retained in a sample retaining device, the method comprising:
 acquiring, by means of an image acquiring device, a plurality of images of said sample at different focus planes within the sample retaining device; and 
 analysing said images, by means of an image analyser, for identifying particles of the sample which are imaged in focus in any one of the analysed images, and for identifying, for each of the identified particles, in which of said analysed images the particle is identified and analysing those particles which have been identified as being imaged in focus in any one of the analysed images using respective image in which respective particle has been identified as being imaged in focus; 
 wherein said plurality of images are acquired at different, essentially parallel, focus planes, which planes are separated from each other by a distance, said distance being less than 10 micrometers; 
 wherein said identification of particles which are imaged in focus is achieved by, for each of the particles: 
 finding an image in which the particle is distinguishable; 
 determining the greatest difference in light intensity between an area of the image which is occupied by the particle and an area of the image where no particle is distinguishable; 
 determining the corresponding differences in light intensity in other images in which the same particle is distinguishable; and 
 identifying the image where the difference in light intensity is the highest; 
 whereby the particle is regarded as being in focus in that identified image. 
 
     
     
       2. The method of  claim 1 , wherein the distance is less than 5 micrometers. 
     
     
       3. The method of  claim 1 , wherein the distance is less than 2 micrometers. 
     
     
       4. The method of  claim 1 , wherein the method further comprises superpositioning the plurality of images, thereby obtaining a superpositioned image containing all the particles imaged in focus at the different focus planes. 
     
     
       5. The method of  claim 1 , wherein only one image per focus plane is acquired. 
     
     
       6. The method of  claim 1 , wherein at least 10 images are acquired. 
     
     
       7. The method of  claim 1 , wherein at least 100 images are acquired. 
     
     
       8. The method of  claim 1 , wherein at least 200 images are acquired. 
     
     
       9. The method of  claim 1 , wherein the sample retaining device is arranged to present the liquid sample for imaging such that the sample has a depth, perpendicular to the focus planes, of at least 100 micrometers. 
     
     
       10. The method of  claim 1 , wherein the sample retaining device is arranged to present the liquid sample for imaging such that the sample has a depth, perpendicular to the focus planes, of at least 200 micrometers. 
     
     
       11. The method of  claim 1 , wherein the sample retaining device is arranged to present the liquid sample for imaging such that the sample has a depth, perpendicular to the focus planes, of at least 500 micrometers. 
     
     
       12. The method of  claim 1 , wherein the sample retaining device is arranged to present the liquid sample for imaging such that the sample has a depth, perpendicular to the focus planes, of 1 millimeter or less. 
     
     
       13. The method of  claim 1 , wherein the liquid sample is a biological sample. 
     
     
       14. The method of  claim 1 , wherein the liquid sample is a blood sample. 
     
     
       15. The method of  claim 1 , wherein the particles are eukaryotic cells. 
     
     
       16. The method of  claim 1 , wherein the particles are mammalian cells. 
     
     
       17. The method of  claim 1 , wherein the particles are human cells. 
     
     
       18. The method of  claim 1 , wherein the particles are bacteria, viruses or platelets. 
     
     
       19. The method of  claim 1 , wherein the particles have a maximum diameter of less than 20 micrometers. 
     
     
       20. The method of  claim 1 , wherein the particles have a maximum diameter of less than 10 micrometers. 
     
     
       21. The method of  claim 1 , wherein the particles have a maximum diameter of less than 2 micrometers. 
     
     
       22. The method of  claim 1 , wherein the analysing of those particles which have been identified as being imaged in focus comprises determining the types and quantities of the particles, the types being distinguished by physical features of the particles, whereby the ratios of different types of particles in the sample are determined. 
     
     
       23. The method of  claim 1 , wherein the particles to be analysed have been stained, by means of a staining agent, prior to the images being acquired. 
     
     
       24. The method of  claim 23 , wherein the liquid sample is contacted with the staining agent, the staining agent being in a dry form, within the sample retaining device, whereby the staining agent is dissolved in the sample. 
     
     
       25. The method of  claim 23 , wherein the staining agent is a fluorescent staining agent. 
     
     
       26. The method of  claim 1 , wherein the image acquiring device is a digital camera. 
     
     
       27. The method of  claim 1 , wherein the images, acquired by means of the image acquiring device, are acquired of a magnification of the liquid sample, said magnification being achieved by means of a light refractor. 
     
     
       28. The method of  claim 1 , wherein the images, acquired by means of the image acquiring device, are acquired of a magnification of the liquid sample, said magnification being achieved by means of a lens. 
     
     
       29. The method of  claim 1 , wherein an imaged volume of the sample is defined by an imaged area of the sample multiplied with a depth of the sample covered by the plurality of images. 
     
     
       30. A method for analysis of particles in a liquid sample, the sample being retained in a sample retaining device, the method comprising:
 acquiring, by means of an image acquiring device, a plurality of images of said sample at different focus planes within the sample retaining device; and 
 analysing said images, by means of an image analyser, for identifying particles of the sample which are imaged in focus in any one of the analysed images, and for identifying, for each of the identified particles, in which of said analysed images the particle is identified and analysing those particles which have been identified as being imaged in focus in any one of the analysed images using respective image in which respective particle has been identified as being imaged in focus; 
 wherein said plurality of images are acquired at different, essentially parallel, focus planes, which planes are separated from each other by a distance, said distance being less than 10 micrometers; 
 wherein said identification of particles which are imaged in focus is achieved by, for each of the particles: 
 finding an image in which the particle is distinguishable; 
 defining an area of that found image, said area comprising the particle and its closest surroundings; 
 determining the pixel variance of that defined area; 
 determining the pixel variance of the corresponding area of other images in which the same particle is distinguishable; and 
 identifying the image where the pixel variance is the highest; 
 whereby the particle is regarded as being in focus in that identified image. 
 
     
     
       31. A measurement apparatus for analysis of particles in a liquid sample, the apparatus comprising an image acquiring device, an image analyser, a holder arranged to hold a sample retaining device retaining the liquid sample, and a light refractor positioned between said image acquiring device and said holder;
 wherein a focus plane is movable stepwise within the sample retaining device when it is held by the holder, whereby the image acquiring device is adapted to acquire a plurality of images of said sample at different focus planes within the sample retaining device, the different focus planes being essentially parallel to each other and being separated from each other by a distance, said distance being less than 10 micrometers; 
 wherein the image analyser is arranged to analyse at least one acquired image for identifying which of the particles are imaged in focus and analysing those particles which have been identified as being imaged in focus; 
 wherein said identification of particles which are imaged in focus is achieved by, for each of the particles: 
 finding an image in which the particle is distinguishable; 
 determining the greatest difference in light intensity between an area of the image which is occupied by the particle and an area of the image where no particle is distinguishable; 
 determining the corresponding differences in light intensity in other images in which the same particle is distinguishable; and 
 identifying the image where the difference in light intensity is the highest; 
 whereby the particle is regarded as being in focus in that identified image. 
 
     
     
       32. The apparatus of  claim 31 , wherein the distance is less than 5 micrometers. 
     
     
       33. The apparatus of  claim 31 , wherein the distance is less than 2 micrometers. 
     
     
       34. A measurement apparatus for analysis of particles in a liquid sample, the apparatus comprising an image acquiring device, an image analyser, a holder arranged to hold a sample retaining device retaining the liquid sample, and a light refractor positioned between said image acquiring device and said holder;
 wherein a focus plane is movable stepwise within the sample retaining device when it is held by the holder, whereby the image acquiring device is adapted to acquire a plurality of images of said sample at different focus planes within the sample retaining device, the different focus planes being essentially parallel to each other and being separated from each other by a distance, said distance being less than 10 micrometers; 
 wherein the image analyser is arranged to analyse at least one acquired image for identifying which of the particles are imaged in focus, and analysing those particles which have been identified as being imaged in focus; 
 wherein said identification of particles which are imaged in focus is achieved by, for each of the particles: 
 finding an image in which the particle is distinguishable; 
 defining an area of that found image, said area comprising the particle and its closest surroundings; 
 determining the pixel variance of that defined area; 
 determining the pixel variance of the corresponding area of other images in which the same particle is distinguishable; and 
 identifying the image where the pixel variance is the highest; 
 whereby the particle is regarded as being in focus in that identified image.